Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus.

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.     

Resuscitation of Vibrio vulnificus from the viable but nonculturable state.

Stationary-phase-grown cells of the estuarine bacterium Vibrio vulnificus became nonculturable in nutrient-limited artificial seawater microcosms after 27 days at 5 degrees C. When the nonculturable cells were subjected to temperature upshift by being placed at room temperature, the original bacterial numbers were detectable by plate counts after 3 days, with a corresponding increase in the direct viable counts from 3% to over 80% of the total cell count. No increase in the total cell count was observed during resuscitation, indicating that the plate count increases were not due to growth of a few culturable cells. Chloramphenicol and ampicillin totally inhibited resuscitation of the nonculturable cells when added to samples that had been at room temperature for up to 24 h. After 72 h of resuscitation, the inhibitors had an easily detectable but reduced effect on the resuscitated cells, indicating that protein and peptidoglycan synthesis were still ongoing. Major changes in the morphology of the cells were discovered. Nonculturable cells of V. vulnificus were small cocci (approximately 1.0 micron in diameter). Upon resuscitation, the cells became large rods with a size of mid-log-phase cells (3.0 microns in length). Four days after the cells had become fully resuscitated, the cell size had decreased to approximately 1.5 micron in length and 0.7 micron in width. The cells were able to go through at least two cycles of nonculturability and subsequent resuscitation without changes in the total cell count. This is the first report of resuscitation, without the addition of nutrient, of nonculturable cells, and it is suggested that temperature may be the determining factor in the resuscitation from this survival, or adaptation, state of certain species in estuarine environments.     

Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state

AIMS: Physiological responses of marine luminous bacteria, Vibrio harveyi (ATCC 14216) and V. fischeri (UM1373) to nutrient-limited normal strength (35 ppt iso-osmolarity) and low (10 ppt hypo-osmolarity) salinity conditions were determined. METHODS AND RESULTS: Plate counts, direct viable counts, actively respiring cell counts, nucleoid-containing cell counts, and total counts were determined. Vibrio harveyi incubated at 22 degrees C in nutrient-limited artificial seawater (ASW) became nonculturable after approximately 62 and 45 d in microcosms of 35 ppt and 10 ppt ASW, respectively. In contrast, V. fischeri became nonculturable at approximately 55 and 31 d in similar microcosms. Recovery of both culturability and luminescence of cells in the viable but nonculturable state was achieved by addition of nutrient broth or nutrient broth supplemented with a carbon source, including luminescence-stimulating compounds. Temperature upshift from 22 degrees C to 30 degrees C or 37 degrees C did not result in recovery from nonculturability. CONCLUSIONS: The study confirms entry of V. harveyi and V. fischeri into the viable but nonculturable state under low-nutrient conditions and demonstrates nutrient-dependent resuscitation from this state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms loss of luminescence of V. harveyi and V. fischeri on entry into the viable but nonculturable state and suggests that enumeration of luminescent cells in water samples may be a rapid method to deduce the nutrient status of a water sample.     

Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment.

Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months. Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, < 15 degrees C). In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca. 21 degrees C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state. These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not. This study indicates that the inability to isolate V. vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift.     

Death of the Escherichia coli K-12 strain W3110 in soil and water.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.     

Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step reaction (30 s [each] at 94 and 65 degrees C) were found to be optimal as well as more time efficient than the three-step PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h. Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene rearrangement or loss of the hemolysin gene, are discussed.     

Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step reaction (30 s [each] at 94 and 65 degrees C) were found to be optimal as well as more time efficient than the three-step PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h. Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene rearrangement or loss of the hemolysin gene, are discussed.     

In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus.

Vibrio vulnificus is an estuarine bacterium responsible for 95% of all seafood-related deaths in the United States. The bacterium occurs naturally in molluscan shellfish, and ingestion of raw oysters is typically the source of human infection. V. vulnificus is also known to enter a viable but nonculturable (VBNC) state, wherein the cells are no longer culturable on routine plating media but can be shown to remain viable. Whether or not this human pathogen remains virulent when entering the VBNC state has not been definitively demonstrated. In this study, the VBNC state was induced through a temperature downshift to 5 degrees C, with cells becoming nonculturable (< 0.1 CFU/ml) within 7 days. As they became nonculturable, virulence was determined by employing an iron overload mouse model. At the point of nonculturability (7 days), injections of the diluted microcosm population resulted in death when < 0.04 CFU was inoculated, although > 10(5) cells in the VBNC state were present in the inoculum. Culturable cells of V. vulnificus, with identification confirmed through PCR, were recovered from the blood and peritoneal cavities of mice which had died from injections of cells present in the VBNC state for at least 3 days. Thus, our data suggest that cells of V. vulnificus remain virulent, at least for some time, when present in the VBNC state and are capable of causing fatal infections following in vivo resuscitation. Our studies also indicate, however, that virulence decreases significantly as cells enter the VBNC state, which may account, at least to some extent, for the decrease in infections caused by this bacterium during winter months.     

Changes in membrane fatty acid composition during entry of Vibrio vulnificus into the viable but nonculturable state

Vibrio vulnificus, a Gram-negative bacterium found in estuarine waters, is responsible for over 95% of all seafood-related deaths in the United States. As a result of a temperature downshift to 5 degrees C, this organism enters the viable but nonculturable (VBNC) state. Changes in the membrane fatty acid (FA) composition of V. vulnificus may be a contributing factor to the ability of this organism to enter into and survive in the VBNC state. This hypothesis was tested by incubating the organism at 5 degrees C in artificial sea water and analyzing the cells' FAs during the initial hours of temperature and nutrient down-shift. Prior to downshift, the predominant FAs were 16:0, 16:1 and 18:0. During the first four hours of downshift, statistically significant changes occurred in 15:0, 16:1, 16:0, 17:0, and 18:0. These results indicate that changes in FA composition occur prior to entry of V. vulnificus into the VBNC state, suggesting that the ability to maintain membrane fluidity may be a factor in this physiological response. Cells in which fatty acid synthesis was inhibited did not survive, indicating that active fatty acid metabolism is essential for entry of cells into the VBNC state.     

Green fluorescent protein-based direct viable count to verify a viable but non-culturable state of Salmonella typhi in environmental samples.

The gfp-tagging method and lux-tagging method were compared to select a better method for verifying a viable but nonculturable (VBNC) state of bacteria in the environment. An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescent protein (GFP). The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi. Using the same method, S. typhi was chromosomally marked with luxAB genes encoding luciferase. The survival of gfp-tagged S. typhi introduced into groundwater microcosms was examined by GFP-based plate count, total cell count, and a direct viable count method. In microcosms containing lux-tagged S. typhi, luminescence-based plate count and the measurement of bioluminescence of each microcosm sample were performed. In microcosms containing lux-tagged S. typhi, viable but nonculturable cells could not be detected by using luminometry. As no distinguishable luminescence signals from the background signals were found in samples containing no culturable cells, a VBNC state of S. typhi could not be verified in lux-based systems. However, comparison between GFP-based direct viable counts and plate counts was a good method for verifying the VBNC state of S. typhi. Because GFP-based direct viable count method provided a direct and precise estimation of viable cells of introduced bacteria into natural environments, it can be used for verifying the VBNC state of bacteria in environmental samples.     

Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate

Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4 degrees C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 10(9) CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.     

Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state.

Entry into the viable but nonculturable state by the human bacterial pathogen Vibrio vulnificus in artificial seawater microcosms was studied. In contrast to the long-term culturability exhibited by cells incubated under these starvation conditions at room temperature, cells exposed to a temperature downshift to 5 degrees C exhibited an immediate decrease in culturability. Cells incubated at low temperature exhibited a morphological change from rods to cocci but demonstrated no reductive division. Of 10 factors studied which might affect the nonculturable response in V. vulnificus, only the physiological age of the cells was found to significantly affect the rate at which cells became nonculturable. The nonculturable response appears to be related to the starvation response, as prestarvation at room temperature for 24 h was found to eliminate the nonculturable response of cells subsequently incubated at 5 degrees C. This observation suggests that the synthesis of starvation proteins may repress the viable but nonculturable program displayed during low-temperature incubation. The possible ecological significance of these findings is discussed.     

Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state.

Entry into the viable but nonculturable state by the human bacterial pathogen Vibrio vulnificus in artificial seawater microcosms was studied. In contrast to the long-term culturability exhibited by cells incubated under these starvation conditions at room temperature, cells exposed to a temperature downshift to 5 degrees C exhibited an immediate decrease in culturability. Cells incubated at low temperature exhibited a morphological change from rods to cocci but demonstrated no reductive division. Of 10 factors studied which might affect the nonculturable response in V. vulnificus, only the physiological age of the cells was found to significantly affect the rate at which cells became nonculturable. The nonculturable response appears to be related to the starvation response, as prestarvation at room temperature for 24 h was found to eliminate the nonculturable response of cells subsequently incubated at 5 degrees C. This observation suggests that the synthesis of starvation proteins may repress the viable but nonculturable program displayed during low-temperature incubation. The possible ecological significance of these findings is discussed.     

Temperature effects on the viable but non-culturable state of Vibrio vulnificus

Abstract The non-culturable state of Vibrio vulnificus , strain C7184, was studied in artificial seawater microcosms held at 5, 10, 15, 20, and 30°C. Plate counts were made on a non-selective medium, total cell counts were monitored by acridine orange epifluorescence, and direct viable counts (DVSs) by the method of Kogure et al. (Can J. Microbiol. 25, 415–420; 1986) and by the INT method. From an initial inoculum of 10⁷ cells/ml, V. vulnificus became non-culturable within 40 days at 5°C, although both indicators of viability revealed a viable population exceeding 10⁶ cells/ml. Cells at all higher temperatures remained culturable (at least 10⁴/ml) throughout the study. The non-culturable states of the opaque and translucent colony variants of V. vulnificus , as well as those of six other clinical and environmental strains of V. vulnificus , were examined at 5°C; all but one strain and both colony variants also became non-culturable within 40 days. In contrast, six other Vibrio spp. ( V. cholerae, V. mimicus, V. parahaemolyticus, V. natriegens, V. proteolyticus , and V. campbelli ) remained culturable at 5°C. Thus, entrance of V. vulnificus into the non-culturable state appears to be highly temperature dependent and, among the vibrios, this species may be especially sensitive to low temperature. The public health aspects of these findings are discussed.     

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4°C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 10⁹ CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment.

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state

The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to "resuscitate" nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.